Project Summary Corneal wound healing is a complex and coordinated process involving injury repair to the epithelial layer and stimulation of limbal stem cell (LSC) proliferation for tissue regeneration. Prevention of excessive stromal myofibroblast activation and vascular ingrowth is also imperative to avoid fibrosis and angiogenesis, which can compromise transparency of the cornea. An approach that can functionally target multiple steps in corneal wound healing may have the potential to significantly improve healing outcomes, leading to novel therapeutic options. This project stems from our novel findings that reveal a vital role for MG53, a tissue repair gene, in modulating LSC function associated with corneal injury-repair. We show that genetic ablation of MG53 leads to injury-induced corneal epithelial thinning, conjunctivalization, stromal fibrosis, and vascularization, all hallmarks of limbal stem cell deficiency (LSCD). Mice with sustained elevation of MG53 in circulation show increased tissue regenerative capacity with enhanced LSC function. MG53 is present in the human tear film, aqueous humor, and corneal epithelial cells, supporting its potential function in corneal homeostasis and wound healing. Using in vivo corneal injury models, we find that MG53 promotes corneal transparency by facilitating epithelial viability and reducing post-injury fibrosis and vascularization. Experiments outlined in this project are centered on testing the hypothesis that MG53 constitutes an active component of the corneal injury- repair and regeneration by maintaining the health of LSCs, as well as controlling the fibrotic response of stromal fibroblasts. We envision that pharmacological formulation to enhance the synergy between MG53 delivery, LSC function, and fibrotic control could be a potentially effective means to treat corneal diseases. The outcome of this research shall have significant translational value in developing potentially effective therapies to treat corneal injury and fibrosis associated with corneal disease.